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Transplantation of low dose CD34⁺KDR⁺ cells promotes vascular and muscular regeneration in ischemic limbs

PAOLO MADEDDU^*,,1, COSTANZA EMANUELI^*,, ELVIRA PELOSI, MARIA BONARIA SALIS^*, ANNA MARIA CERIO, GIUSEPPINA BONANNO, MARIELLA PATTI, GIORGIO STASSI, GIANLUIGI CONDORELLI^||, and CESARE PESCHLE^,,1

Thomas Jefferson University, Philadelphia, Pennsylvania, USA;
^* Experimental Medicine and Gene Therapy, INBB, Osilo;
Biotechnology and Molecular Medicine, INBB, Pula;
Department of Internal Medicine, Sassari University;
Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome;
Department of Surgical and Oncological Science, University of Palermo;
Department of Obstetrics and Gynecology, Catholic University, Rome; and
^|| Laboratory of Molecular Cardiology, San Raffaele Biomedical Science Park, Rome, Italy

¹Correspondence: C.P., Kimmel Cancer Institute, Thomas Jefferson University, Room #609, 233 South 10th St., Philadelphia, PA 19107-5541, USA. E-mail: cesare.peschle{at}mail.tju.edu or P.M., Experimental Medicine and Gene Therapy Unit, National Institute of Biostructures and Biosystems (INBB), Via S. Antonio, 07033 Osilo (SS). E-mail: madeddu{at}yahoo.com

SPECIFIC AIMS

The optimal cell population to be transplanted for therapeutic revascularization of ischemic tissues remains undetermined. We have compared the therapeutic potential of two subsets of human cord blood CD34⁺ progenitors that express or do not express the VEGF-A receptor 2 (KDR). As major treatment end-points, we considered 1) the level of hemodynamic recovery from ischemia, 2) histological evidence of microvascular and skeletal myofiber regeneration, and 3) protection from ischemia-induced apoptosis and replacement fibrosis.

PRINCIPAL FINDINGS

1. CD34⁺KDR⁺ cell transplantation enhances the neovascularization response to ischemia and protects from apoptosis
The proangiogenic action of transplanted CD34⁺KDR⁺ cells was tested in immunodeficient mice submitted to operative unilateral limb ischemia. As shown in Fig. 1 A, transplantation of 10³ CD34⁺KDR⁺ cells increased capillary density of ischemic muscles by 114% (1408±96 vs. 659±28 cap/mm² in contralateral normoperfused adductors, P<0.01). No further increase in capillarity was observed by doubling the cell dosage. The angiogenic effect promoted by CD34⁺KDR⁺ cells largely exceeded (P<0.01) the increment seen in muscles injected with vehicle (66%) or 10⁴ CD34⁺KDR^– cells (46%).

View larger version (21K): [in this window] [in a new window]   Figure 1. A) Capillary density in ischemic adductor muscles and contralateral normoperfused ones (open columns). Mice underwent surgical excision of left femoral artery and had ischemic muscles injected with vehicle, cord blood-derived CD34+KDR– (104) or CD34+KDR+ (103 or 2x103) cells. Capillary density of ischemic (filled columns) and contralateral adductor skeletal muscles (open columns) was counted 21 days after induction of ischemia. Sample number is indicated within columns and cell dosage is displayed on the abscissa. Values are mean ±SE. *P <0.05 vs. contralateral; +P < 0.05 vs. vehicle and vs. CD34+KDR–. B) Arteriole density in adductor muscles subjected to ischemia (filled columns) and contralateral ones (open columns). Values are mean ±SE. *P < 0.05 vs. contralateral; +P < 0.05 vs. vehicle and vs. CD34+KDR–.

Figure 1B shows that arteriogenesis was potentiated by transplantation of CD34⁺KDR⁺ cells. This effect was dosage dependent: in fact, 10³ CD34⁺KDR⁺ cells did not augment the spontaneous increase in arteriole density whereas 2 x 10³ cells potently stimulated arteriogenesis. In contrast, arteriole density of muscle injected with 10⁴ CD34⁺KDR^– cells did not differ from that of controls (P=N.S.).

Human nuclear antigen (HNAg) -positive endothelial cells (ECs) were revealed in capillaries from CD34⁺KDR⁺ transplanted muscles with greater frequency (9.0±1.2/2000 capillaries) compared with the CD34⁺KDR^– group (2.1±0.2, P<0.01). Similarly, immunohistochemical evidence indicates increased incorporation of CD34⁺KDR⁺ cells into arteriole endothelium.

Abundant expression of Ki67, a proliferation-associated protein, was detected in differentiated HNAg⁺ ECs, suggesting that CD34⁺KDR⁺-derived cells continuously contribute to reparative angiogenesis up to late stages of the healing process.

We evaluated the effects of cell therapy on ischemia-induced apoptosis. TUNEL-positive ECs were significantly less in CD34⁺KDR⁺ injected adductors (3.8±0.9 cells/1000 cap) than vehicle- or CD34⁺KDR^–-injected ones (9.5±2.5 and 8.4±1.6 cells/1000 cap, P <0.05 for both comparisons). Myofiber apoptosis was reduced by CD34⁺KDR⁺ transplantation (P<0.01).

3. CD34⁺KDR⁺ cell transplantation prevents fibrosis and promotes myofiber regeneration
We further analyzed the morphology of limb adductor muscles stained with Azan Mallory. Interstitial fibrosis was increased in vehicle-injected ischemic muscles (54±3% of total section area) compared with normoperfused contralateral ones (3±1%, P<0.01). Transplantation of CD34⁺KDR⁺ cells resulted in a marked attenuation of fibrosis (10±2%, P<0.01 vs. vehicle); the antifibrotic effect of CD34⁺KDR^– cells, transplanted at a 1-log more elevated number, was significantly less pronounced (30±1%, P<0.01 vs. CD34⁺KDR⁺).

To determine the exact location of CB cell incorporation, we performed high-resolution, confocal analysis of skeletal muscle sections stained with antibodies raised against HNAg, dystrophin (a marker for sarcolemma), laminin (labeling basal lamina), or desmin (a marker for interstitial cells). In muscles injected with CB cells, donor human nuclei were identified underneath the sarcolemma and the basal lamina, as well as in interstitial space. The results of cell counting performed in 3500 skeletal myocytes indicate an increased incorporation of CD34⁺KDR⁺ cells vs. CD34⁺KDR^– cells into the myofibers (12.1±0.7 and 6.0±0.5, respectively, P<0.01) and underneath the basal lamina (8.0±0.9 and 4.0±0.7, P<0.01); no group difference was observed with regard to incorporation into cells of the interstitial space (15.1±0.5 and 12.3±0.6, P=N.S.).

4. CD34⁺KDR⁺ cell transplantation improves spontaneous hemodynamic recovery and clinical outcome.
In the vehicle group, 13 (75%) of 18 mice underwent spontaneous foot autoamputation; the remainder showed an ischemic to contralateral blood flow ratio of 0.27 ± 0.03 at 21 days. Clinical outcome remained unaffected by transplantation of 10⁴ CD34⁺KDR^– cells: 7 (50%) of 14 underwent autoamputation, with the remainder showing ischemic to contralateral ratio of 0.28 ± 0.04 (P=N.S. vs. vehicle). In contrast, 2 x 10³ CD34⁺KDR⁺ cell transplantation resulted in improved outcome as denoted by reduced autoamputation rate (15% of 14 mice, P<0.05 vs. vehicle or CD34⁺KDR^–). In the remainders, hemodynamic recovery was ameliorated by CD34⁺KDR⁺ cell transplantation (denoted by an ischemic to contralateral ratio of 0.40±0.05, P<0.05 vs. vehicle-treated or CD34⁺KDR^–). Hemodynamic recovery improved in mice transplanted with 2 x 10³ CD34⁺KDR⁺ cells. A lower dosage (10³ cells) produced a milder improvement of clinical outcome (data not shown).

CONCLUSIONS AND SIGNIFICANCE

We observed that in a murine model of limb ischemia, transplantation of a low number of human CB CD34⁺KDR⁺ cells markedly stimulates spontaneous capillarization and arteriogenesis. Conversely, transplantation of a larger number of CD34⁺KDR^– cells is largely ineffective. Improvement of neovascularization might be attributed to 1) differentiation of progenitor cells into ECs, which maintain a proliferative phenotype instrumental to healing, and/or 2) release of endothelial GFs by transplanted cells. In view of the low level of incorporation of human cells in the generated vessels, we suggest that the transplanted EPCs may exert significant trophic effects on EC proliferation and viability via GF release (Fig. 2 ). This mechanism is supported by the observation that cultured CD34⁺KDR⁺ cells show a unique capacity to resist to serum starvation and to release relatively large amounts of VEGF-A. In agreement with a trophic mechanism is the observation that CD34⁺KDR⁺ cell transplantation hinders apoptotic cell death and concomitantly reduces fibrosis in ischemic muscles.

View larger version (29K): [in this window] [in a new window]   Figure 2. The present study examined the regenerative potential of two subpopulations of CD34+ progenitor cells from human cord blood, selected for the ability to express the VEGF receptor 2 (KDR). The CD34+KDR+ subset comprises endothelial precursor cells (EPCs), primitive hematopoietic cells, and hemangioblasts; the CD34+KDR– subfraction largely consists of hematopoietic progenitor cells (HPCs). Top: In serum-free starvation culture, CD34+KDR+ cells release larger amounts of VEGF and are markedly less susceptible to apoptosis compared with their CD34+KDR– counterparts. Bottom: The regenerative potential of these 2 subpopulations was compared in vivo. A) 103 or 2 x 103 CD34+KDR+ vs. 104 CD34+KDR– cells were transplanted into the ischemic adductors of immunodeficient mice: transplantation was performed at induction of ischemia by surgical excision of the left femoral artery. B–F) Suggested fate of the transplanted EPCs and therapeutic mechanisms. B) CD34+KDR+ (but not KDR–) cells are resistant to apoptosis when transplanted in the ischemic limb; C) the CD34+KDR+ subfraction may proliferate in that it is enriched for not only EPCs, but also for hemangioblasts endowed with self-renewal activity; D) the release of VEGF-A by CD34+KDR+ cells exerts a significant anti-apoptotic and proliferative action on murine Flk1+ ECs in the transplanted area; E) CD34+KDR+ cells incorporate in nascent capillaries and arterioles and differentiate into mature human ECs; F) CD34+KDR+ cells may exert a trophic action on murine skeletal myofibers and contribute to their regeneration directly by trans-differentiation and/or cell fusion. Through these mechanisms, CD34+KDR+ cells foster neoangiogenesis and thereby accelerate limb hemodynamic recovery and healing.

We observed that CD34⁺KDR⁺ cell transplantation significantly stimulates muscle regeneration. This may derive from the improved reperfusion of the injured limb. CD34⁺KDR⁺ progenitors/stem cells might have contributed directly to myogenic regeneration and/or cell fusion. More important, the present study documents that injection of CD34⁺KDR⁺ cells improves the clinical outcome of ischemic muscles and accelerates the rate of hemodynamic recovery. These results indicate that the microvasculature generated with the possible contribution of CD34⁺KDR⁺ cells was functionally efficient.

The therapeutic efficacy of a small number of CD34⁺KDR⁺ cells is a novel that compares favorably with previous studies using markedly more elevated number of unselected progenitor cells. The therapeutic efficacy of low dose CD34⁺KDR⁺ cells is seemingly mediated by diverse factors. 1) The CD34⁺KDR⁺ subfraction is enriched for EPCs/HSCs and comprises hemangioblasts. Conversely, CD34⁺KDR^– cells are largely constituted by HPCs, while they are not enriched for EPCs/HSCs and do not contain hemangioblasts. It is not surprising, therefore, that the CD34⁺KDR⁺ cells transplanted in the ischemic area generate human ECs and perhaps skeletal muscle cell, whereas the CD34⁺KDR^– subfraction has a markedly lower cell generation capacity. 2) CD34⁺KDR⁺ cells are exquisitely resistant to serum starvation; conversely, CD34⁺KDR^– cells rapidly die in these harsh culture conditions. Similarly, CD34⁺KDR⁺ (but not KDR^–) cells may be resistant to apoptosis when transplanted in the ischemic limb. 3) The release of relatively large amounts of VEGF-A by a small number of CD34⁺KDR⁺ cells might exert significant anti-apoptotic and proliferative action on ECs in the transplanted area.

The CD34⁺KDR⁺ transplantation approach triggers important questions. 1) The CD34⁺KDR⁺ subset comprises not only EPCs, but also primitive hematopoietic cells and hemangioblasts. The relative contribution of these functional cell subsets to the diverse ameliorative effects after CD34⁺KDR⁺ cell transplantation in the ischemic limb is not clear. However, these functionally different cell populations may represent a single cell pool exhibiting hematopoietic and/or endothelial differentiation in different microenvironmental conditions. 2) The number of available CD34⁺KDR⁺ cells is limited. Ongoing studies in our laboratory aim to expand ex vivo the CD34⁺KDR⁺ subset to facilitate its therapeutic use at preclinical and then possibly clinical level.

We propose that the CD34⁺KDR⁺ cell population may be regarded as a potentially interesting tool for regenerative therapy in limb ischemia, particularly if coupled with a preliminary ex vivo expansion step inducing primitive cell self-renewal.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2192fje

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